In the last 10 to 15 years, two methods for the soft ionization of biological macromolecules have prevailed in mass spectrometric analysis: matrix-assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI). The biological macromolecules to be analyzed are termed analyte molecules below. With the MALDI method, the analyte molecules are generally prepared in a solid matrix on the surface of a sample support, whereas with the ESI method they are dissolved in a liquid. Both methods have a considerable influence on the mass spectrometric analysis of biological macromolecules in genomics, proteomics and metabolomics; their inventors were awarded the Nobel prize for chemistry in 2002.
In a prepared MALDI sample, there are 103 to 105 times as many matrix molecules as there are analyte molecules, and they form a polycrystalline matrix in which the analyte molecules are embedded, isolated in the interior of the crystals or at their grain boundaries. The prepared MALDI sample is irradiated with a short-time laser pulse, which is strongly absorbed by the matrix molecules. The pulsed laser irradiation means that the matrix is explosively transferred from the solid state into the gaseous phase of a vaporization cloud (desorption). The analyte molecules are usually ionized by being protonated or deprotonated in reactions with matrix molecules or matrix ions, the analyte ions being predominantly singly charged after leaving the vaporization cloud. The degree of ionization of the analyte molecules is only some 10−4. The term soft ionization is used when an analyte molecule is transferred separately into the gaseous phase and ionized without undergoing any bond breakage.
Despite the linear absorption by the matrix, matrix-assisted laser desorption/ionization is a nonlinear process, which for pulsed laser radiation with a duration of a few nanoseconds only starts above an intensity threshold of around 106 watts per square centimeter. For soft ionization, the maximum intensity lies at an upper limit of approximately 107 watts per square centimeter. With a typical duration of around ten nanoseconds, the stated intensity limits result in a fluence of the laser radiation between 10 and 100 millijoules per square centimeter.
The MALDI process is complex and affected by numerous factors, some of which are interdependent. Since the MALDI method was first published in 1988, many parameters have been investigated and varied. In spite of this, the processes in the matrix and in the vaporization cloud, which lead to the ionization of the analyte molecules, are still not completely understood and are still under intense research (K. Dreisewerd, Chem Rev. 103 (2003), 395-425: “The Desorption Process in MALDI”).
The chemical parameters of the MALDI process, for example the matrix substances themselves, the concentration ratio between matrix and analyte molecules, and the preparation conditions, have been comprehensively researched. For analyte molecules of different chemical substance classes. such as proteins or nucleic acids, over one hundred different chemical matrix substances are known, such as sinapic acid, DHB (2,5-dihydroxy benzoic acid), CHCA (α-cyano-4-hydroxy cinnamic acid) or HPA (3-hydroxypicolinic acid). The matrix substances exhibit strong absorption in the wavelength range between 330 and 360 nanometers. A MALDI sample can be prepared in a number of different ways, for example with “dried droplet” preparation or thin layer preparation. In “dried droplet” preparation, the matrix substance is dissolved together with the analyte molecules in a solvent, applied to a sample support, and then dried slowly in air. In thin layer preparation, on the other hand, the matrix substance without analyte molecules is dissolved in a volatile solvent such as acetone or acetonitrile, and applied to the sample support. Compared with “dried droplet” preparation, the volatile solvent evaporates very quickly and facilitates the creation of a thin, homogeneous matrix layer. A solution with analyte molecules is then applied to the thin matrix layer, causing the latter to be partially dissolved again, and the analyte molecules are integrated into the matrix during the subsequent drying. Whereas in thin layer preparation a homogeneous MALDI sample with microcrystals is produced, in “dried droplet” preparation larger crystals are formed and the surface of the MALDI sample shows a distinct morphology with different sample thicknesses.
As far as the physical parameters of the MALDI process are concerned, until now the temporal duration of the laser pulses, the intensity in the laser focus, and the wavelength of the pulsed laser radiation have chiefly been considered.
Nowadays, commercially available mass spectrometers with MALDI mainly use pulsed laser systems in the ultraviolet spectral range (UV). A number of laser types and wavelengths are available: nitrogen laser (λ=337 nm), excimer lasers (λ=193 nm, 248 nm, 308 nm), Nd:YLF laser (λ=349 nm), and Nd:YAG laser (λ=266 nm, 355 nm). Only the nitrogen laser and the Nd:YAG laser at a wavelength of 355 nanometers are of commercial interest for the MALDI method far and away the most frequently used. The laser medium of the nitrogen laser is a gas, whereas with the Nd:YAG laser it is a YAG (yttrium aluminium garnet: Y3Al5O12) crystal doped with neodymium ions. With the Nd:YAG laser, the strongest laser line, at a wavelength of 1064 nanometers, is turned into the stated wavelengths in nonlinear optical crystals. The duration of the laser pulses used in the MALDI method is typically between 1 and 20 nanoseconds in the UV. In the academic field, however, pulse durations in the region of picoseconds have also been used.
For the MALDI method, laser systems which emit in the infrared spectral region (IR): Er:YAG (λ=2.94 μm) and CO2 (λ=10.6 μm) are also occasionally used in the field of research. Whereas with the UV-MALDI method the matrix molecules are supplied with energy via excited electronic states, in the IR-MALDI method molecular oscillations of the matrix molecules are excited. The pulse duration of the IR laser systems in the IR-MALDI method are between 6 and 200 nanoseconds. In contrast to the UV-MALDI method, both solid matrices and liquid matrices, for example glycerine, are used in the IR-MALDI method.
The laser systems used in the MALDI method differ not only in their wavelength but also in their spatial beam profile. For solid-state lasers such as the Nd:YAG laser or the Er:YAG laser, the laser medium is a crystal doped with ions. The laser medium is located in an optical resonator, which ensures that the spatial beam profile consists of one transverse fundamental mode or a few transverse beam modes. The radial intensity distribution of the transverse fundamental mode corresponds to a Gaussian function and is rotationally symmetric to the direction of propagation of the laser radiation. A laser beam like this can be focused to a minimum diameter which is limited only by the diffraction.
The nitrogen laser at a wavelength of 337 nanometers is by far the most frequently used type of laser in the MALDI method, this wavelength being the most intensive laser line of the nitrogen laser. The laser medium used is gaseous nitrogen, which is excited by means of an electrical discharge between two electrodes elongated along the beam direction. Since the most intensive laser line exhibits a high amplification, a laser pulse can remove the population inversion of the energy states even if it passes along the electrodes only once. Even when using a resonator with mirrors, many transverse beam modes are superimposed in the beam profile of the nitrogen laser, with the result that the minimum diameter of a laser focus in commercial nitrogen lasers at a wavelength of 337 nanometers is only around three micrometers. The typical diameter of the area irradiated in MALDI applications is around 20 to 200 micrometers. The beam profile has a rectangular shape at the front side of the two electrodes, the geometrical dimensions of the beam profile being determined by the width and spacing of the discharge electrodes. The intensity distribution inside of the rectangular shape is approximately homogenous (i.e. flat-top beam profile). The repetition rate of the laser pulses in the nitrogen laser is limited to around 100 hertz unless provision is made for a rapid gas exchange. Nitrogen lasers with a typical repetition rate of 50 hertz are used for MALDI applications.
In practice, the electrical gas discharge in the nitrogen laser is not the same pointing the discharge volume between the electrodes generating a spatially inhomogeneous amplification profile. The inhomogeneous amplification does not even out during the short time the laser is in action, but instead transfers to the intensity distribution of the beam profile of the nitrogen laser. The nitrogen laser thus has a spatially modulated flat-top beam profile with intensity maxima and minima, which is imaged onto the sample or focused onto it. These inhomogeneities being inherently present in the beam profile of the nitrogen laser lead to an intensity distribution of the laser radiation on the sample being spatially modulated and always exhibiting a multiplicity of intensity peaks.
The pulsed solid-state lasers used until now in the MALDI method usually have a beam profile which comes very close to a single Gaussian beam mode. If a pulsed laser beam is focused or imaged onto the sample, then at the location of the sample there is a Gaussian intensity distribution with a single intensity peak. The width of an intensity peak is generally given by the so-called half-width. In the region of the half-width, the intensity is greater than half the maximum intensity of the intensity peak. With solid-state lasers in the UV, the half-width can theoretically be less than one micrometer, but in MALDI applications it is typically between 20 and 200 micrometers. Even if laser pulse repetition rates of several hundred kilohertz are possible in principle with solid-state lasers, most current MALDI applications operate with a repetition rate of up to 200 hertz. The energy fluctuations from laser pulse to laser pulse are typically smaller in the case of solid-state lasers than with nitrogen lasers.
According to the prior art, the attempt is often made to achieve a spatially homogeneous intensity distribution on the sample in order to even out the inhomogeneities of the prepared MALDI sample, which occur, for example, in the case of non-uniform embedding of the analyte molecules in the matrix. In order to obtain a homogeneous intensity distribution on the sample with a Gaussian beam profile of a solid-state laser, the beam profile can be spatially homogenized by propagation in a fiber, and then imaged onto the sample. To facilitate this, the laser beam is coupled into a fiber in which a large number of transverse fiber modes with differing radial intensity distributions can propagate (multimode fibers). The propagation of the coupled laser beam in the multimode fiber means that energy is transferred out of the Gaussian beam mode and into a large number of transverse fiber modes which are superimposed at the output of the fiber. If the temporal coherence of the laser radiation used is sufficiently low, or the multimode fiber is sufficiently long, the intensity distribution at the end of the fiber is given by the sum of the intensity distributions of the individual transverse fiber modes. The large number of transverse fiber modes with differing radial intensity profiles thus results in a homogeneous intensity distribution at the end of the fiber. If the end of the multimode fiber is now imaged, a flat-top intensity distribution is also obtained on the sample. This method of homogenizing the beam profile is also used with the nitrogen laser to minimize the inherent inhomogeneities in the beam profile.
The quality of a mass spectrometric analysis is generally determined by the following parameters: mass accuracy, mass resolution, detection power, quantitative reproducibility and signal-to-noise ratio. The quality of a mass spectrometric analysis increases if at least one parameter is improved and the other parameters do not deteriorate as a result. The mass accuracy includes both a systematic deviation of the measured average ion mass from the true ion mass (mass trueness, or rather the deviation from mass trueness) and the statistical variance of the individual measured values around the mean of the ion mass (mass precision). The mass resolution determines which ion masses in the mass spectrometric analysis can still be distinguished. In practice, however, it is not only the quality but also the robustness of the mass spectrometric analysis that is important. A mass spectrometric analysis is robust if its quality changes little when the measuring parameters, for example the energy of the laser pulses or the preparation conditions of the MALDI sample, are
The ion signal of a mass spectrometer with MALDI is proportional to the ionization efficiency, to the desorbed sample volume and to the concentration of the analyte molecules in the sample. The ionization efficiency is given by the number of analyte ions, which can be evaluated, divided by the number of analyte molecules in the desorbed sample volume, i.e., the percentage of analyte molecules from the sample volume ablated by the laser irradiation which are available as ions for a mass spectrometric analysis. If analyte molecules are already present in the matrix as ions before the desorption process, the number of analyte molecules is increased by the number of analyte ions being already ionized. Since the desorbed sample volume can be relatively easily increased by the irradiated sample area and by the fluence, the ionization efficiency represents an important parameter for the optimization of the MALDI process. A high ionization efficiency permits a high detection power because a maximum ion signal at low concentration (or at low sample consumption) is achieved. With a typical degree of ionization of only 10−4 it is possible to considerably improve the MALDI process. The definition of the ionization efficiency of the MALDI process also takes into account the losses which arise as a result of a fragmentation of analyte molecules during the transfer into the gaseous phase, and therefore reduce the number of analyte ions which can be evaluated.
For mass spectrometric analysis of the analyte ions generated in the MALDI process, conventional sector field mass spectrometers and quadrupole mass spectrometers are suitable in principle, as are quadrupole ion trap mass spectrometers and ion cyclotron resonance mass spectrometers. However, particularly suitable are time-of-flight mass spectrometers with axial injection, which require a pulsed current of ions to measure the time of flight (TOF). In this case, the starting point for the time of flight measurement is dictated by the ionizing laser pulse. The MALDI process was originally developed for use in a vacuum. In more recent developments, matrix-assisted laser desorption/ionization is also used at atmospheric pressure (AP MALDI) or intermediate pressure. Here, the ions are generated with a repetition rate of up to 2 kilohertz and fed, with the help of an ion guide, to a with orthogonal injection (OTOF “orthogonal time of flight”), a quadrupole ion trap mass spectrometer or an ion cyclotron resonance mass spectrometer. In an OTOF mass spectrometer, the ions generated in the MALDI process can be fragmented and stored before the measurement of the time of flight is started by an electronic pulsed injection.
With specific analytical methods, the intensity on the sample is increased to such a degree that the ions generated have enough intrinsic energy to dissociate. Depending on the time between the generation of the ions and their dissociation, this is termed a decay within the ion source (ISD or “in-source decay”) or outside the ion source (PSD or “post-source decay”).
Moreover, there are also methods of imaging mass spectrometry analysis (IMS) in which the MALDI process is used to generate the ions. With IMS, a thin section of tissue obtained, for example, from a human organ, using a microtome, is prepared with a matrix substance, and mass spectrometrically analyzed with spatial resolution. The spatial resolution of the mass spectrometric analysis can be done either by scanning individual small spots of the tissue section or by stigmatic imaging of the ions generated. With the scanning method, the pulsed laser beam is focused onto a small diameter on the sample, and a mass spectrum is measured for each individual pixel. A one- or two-dimensional frequency distribution is produced for individual proteins from the large number of individual spatially resolved mass spectra. With stigmatic imaging, an area of up to 200 by 200 micrometers is irradiated homogeneously with a laser pulse. The ions generated in this way are imaged pixel by pixel onto a spatially resolving detector by an ion optic. Until now it has only been possible to scan the frequency distribution of one ion mass with a single laser pulse because spatially resolving ion detectors that operate fast enough are not available. The measured ion mass can be varied from laser pulse to laser pulse, however.